Hospitalized patients are at high risk for developing infections caused by multidrug-resistant (MDR) bacteria. According to the Infectious Diseases Society of America MDR Gram-negative pathogens such as P. aeruginosa, A. baumannii and extended-spectrum b-lactamase producing Enterobacteriaceae are especially problematic with high attributable mortality rates, and unique virulence or drug-resistance factors. Infections caused by these pathogens are exceedingly difficult to treat due antibiotic resistance and formation of biofilms that are resistant to host defense and antimicrobial treatments. Bacterial biofilms are a key challenge in treating chronic wound infections. Chronic wounds are particularly susceptible to the development of infections and bacterial biofilms are implicated in both the infection of wounds and failure of those wounds to heal. Given the severity of the problem, there is an undeniable and urgent need for new antibiotics active against MDR Gram-negative pathogens and capable of modulating bacterial biofilm formation and maintenance To address the need for the discovery of novel antibacterial agents for topical application and effective against MDR Gram-negative bacteria, including their biofilms, we propose to design and synthesize novel cyclic lipopeptides based on fusaricidin/LI-F natural products. Our recent breakthrough finding have shown that that the increase in overall hydrophobicity and net positive charge of these cyclic lipopeptides improved their activity against Gram-negative bacteria. The improvement in potency is particularly evident in the activity against bacterial biofilms, as the lead peptide 3 showed promising in vitro and in vivo activities against P. aeruginosa and A. baumannii biofilms. Importantly, structural changes resulting in more hydrophobic and positively charged analogues did not lead to increased nonspecific toxicity. Within this proposal, our overall goal is to further modify cyclic lipopeptide 3 to optimize its antibacterial/antibiofilm activity and to minimize nonspecific toxicity. Within the R21 phase we will utilize combinatorial chemistry in conjunction with screening assays to identify cyclic lipopeptide leads with high potency against planktonic and biofilm MDR Gram-negative bacteria and low nonspecific toxicity. In the R33 phase we will: a) develop an optimal delivery system for topical application based on gelatin hydrogels, b) determine the mode of action of these peptides, and c) assess the therapeutic potential of an optimized peptide/hydrogel formulation in a porcine wound infection model. Once completed, the proposed research will lead to a new class of antibacterial peptides for topical application that can be advanced to clinical studies. Once completed, the proposed research will lead to a new class of antibacterial peptides for topical application that can be advanced to clinical studies.